Coding

Part:BBa_K1499001

Designed by: Jotthe Kannappan   Group: iGEM14_StanfordBrownSpelman   (2014-08-27)

wssG gene

This part encodes wssG, a gene found in the wss operon of Pseudomonas fluorescens theorized to be involved in the acetylation of cellulose.

Usage and Biology

wssG is one of the four genes isolated from Pseudomonas fluorescens responsible for acetylating cellulose (the others being wssFHI, K149000, 2, 3). The wss operon in P. fluorescens contains 10 genes responsible for biofilm production, allowing the bacterium to colonize the air-liquid interface. The operon includes genes necessary for the biosynthesis of cellulose (including many with homologies to E. coli or G. hansenii, including minD, subunits of cellulose synthase, and a cellulose), and four (wssF-I) have been implicated as responsible for the acetylation of the cellulose polymer. wssG has been purported to form an acetylation complex associated with cellulose synthase [1].

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 570
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

Verification

We successful isolated the four acetylation genes from P. fluorescens total DNA (Figure 1).

Figure 1. Pure genomic DNA was isolated from P. fluorescens and the four acetylation genes were amplified

The part sequence contained BioBrick restriction sites, which had to be removed before sequencing (see Design Page). The part was sequence verified after insertion into pSB1C3 and before submission to the registry with two reads using VF2 and VR (Figure 2).

Figure 2. Sequencing alignment showing the proper insertion of wssG into pSB1C3. The first sequence disagreement is a substitution mutation we accounted for being unlikely to affect protein function. The second confirms that mutagenesis worked and removed the EcoRI site.

Results

This part has been successfully cloned into the BioBrick backbone, but for expression in G. hansenii, our model cellulose-producing organism, we needed to use the multi-host shuttle vector pUCD4 (Figure 3).

Figure 3. Multi-host shuttle vector pUCD4

This procedure was more time-consuming than expected, as the pUCD4 plasmid is >10kb, the maximum size able to be purified by spin columns. Further work is needed to prove the activity of the wss genes in acetylating cellulose.

References

[1] Spiers AJ et al. (2013) Cellulose Expression in Pseudomonas fluorescens SBW25 and Other Environmental Pseudomonads in Cellulose - Medical, Pharmaceutical, and Electronic Applications. DOI: 10.5772/53736

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